The phosphorylation of myosin light chains and heavy chains by protein kinase C is known to be temporally correlated with Ca2+-dependent secretion of granules from RBL-2H3 cells. We have now shown that whereas myosin light chains are predominantly monophosphorylated by the Ca2+/calmodulin-dependent myosin light chain kinase at serine 19 in unstimulated cells, stimulation of RBL-2H3 cells with antigen or other secretagogues causes an additional phosphorylation of myosin light chains by myosin light chain kinase at threonine 18, as well as by protein kinase C at serine 1 or serine 2. This diphosphorylation at serine 19 and threonine 18 by myosin light chain kinase and the monophosphorylation by protein kinase C is correlated with the rate and extent of degranulation. Other experiments supported the idea that these phosphorylations were closely associated with exocytosis in RBL-2H3 cells. Thus, phosphorylation, as well as secretion, can be blocked by the kinase inhibitors KT5926 and ML-7. More specifically, phorbol ester alone induces phosphorylation of myosin light chains by protein kinase C exclusively, but fails to induce secretion until accompanied by low concentrations of A23187, which activates myosin light chain kinase. Conversely, selective suppression of phosphorylation by protein kinase C (with Ro7549 in antigen-stimulated cells) suppresses degranulation, thereby indicating a requirement for protein kinase C.